Jan-Willem de Gier Center for Biomembrane Research, Stockholm, Sweden Green Light for Membrane Proteins.

Slides:

Advertisements

Similar presentations

Today’s lecture (Based on Chapter 1): 1. Basic organization of the cell 2. The static cell vs the living cell 3. Experimental approaches in cell physiology.

Advertisements

High-throughput, flourescent-based optimisation of eukaryotic membrane protein overexpression and purification. Simon Newstead, Joy Kim, Gunnar von Heijne,

Application in Molecular Cloning David Shiuan Department of Life Science, Institute of Biotechnology and Interdisciplinary Program of Bioinformatics National.

Bacillus subtilis expression/secretion systems

Research In the service of humanity, for the society of tomorrow.

Single Cell Variability The contribution of noise to biological systems.

Cytological screening for novel cell division genes in Escherichia coli Florian Szardenings Final Year Project Gerdes Lab, 2nd Floor Cookson Building Institute.

Skin temperature, tactile processing, and vividness of the illusion Moseley G L et al. PNAS 2008;105: ©2008 by National Academy of Sciences.

Functional and Evolutionary Attributes through Analysis of Metabolism Sophia Tsoka European Bioinformatics Institute Cambridge UK.

Aberrant Wnt /  -catenin signaling can induce chromosomal instability in colon cancer Behrens. J et al, PNAS, 2006.

GFP-based membrane protein overexpression and purification in E. coli and S. cerevisiae Joy Kim Center for Biomembrane Research Department of Biochemistry.

1 Genomics Advances in 1990 ’ s Gene –Expressed sequence tag (EST) –Sequence database Information –Public accessible –Browser-based, user-friendly bioinformatics.

GFP‐Sed5p localization defect in sgt2Δ.

Yeast Two-Hybrid Screening: The Principles

Figure 1 Histopathology of low-grade glioma

Yeast Tgl3p expressed in HeLa cells localizes to lipid droplets.

Melting behavior of proteins identified in the Escherichia coli meltome and their properties Melting behavior of proteins identified in the Escherichia.

Overview of the experimental setting.

Fig. 1. Generation of ERY974. Generation of ERY974. (A) Schematic illustration of ERY974 structure and the introduced mutations. The two Fab arms share.

Phosphorylation and sequence disorder in microtubule-associated protein Tau.A, schematic illustration of the domain profile of Tau with all known phosphorylation.

Total funding for cerebral palsy research by National Health and Medical Research Council (NHMRC, Australia), Cerebral Palsy Alliance Research Foundation.

Volume 49, Issue 1, Pages (January 2013)

Percentage of proteins identified in envelope membrane extracts according to the purification method and the number of transmembrane domains. Percentage.

Differential conjugation of endogenous SUMO-1 and endogenous SUMO-2/3 to target proteins.A and B, SUMO-1 and SUMO-2 proteins were produced in E coli and.

Frequency distribution of the GRAVY of the theoretical proteins (open bars) and of 110 genes encoding proteins identified on a 2-D electrophoresis gel,

Volume 16, Issue 1, Pages (June 2016)

Distributions of the ELDP values and Mascot scores for all protein identifications.a, frequency of ELDP value returned by correct (gray bars) and incorrect.

Relative quantification of differentially expressed membrane proteins, distribution of coefficients of variation, and performance of the respiratory chain.A,

Analysis of growth, protein expression, and morphology

2D BN-PAGE methodology suitable for relative quantification and 2D BN-PAGE reference map of the E. coli cytoplasmic membrane proteome.A, to be able to.

Experimental setup.A, topology models of the overexpressed membrane protein GFP fusions. Experimental setup.A, topology models of the overexpressed membrane.

Membrane protein overexpression affects the pH of the culture and protein secretion.A, the pH of the culture of GFP fusion-overexpressing cells and the.

Mass spectrometry identification of spots in 2D blue native gels of cytoplasmic membranes isolated from BL21(DE3)pLysS Spots in 2D BN gels of cytoplasmic.

Relative quantification of the cytoplasmic membrane proteome by 2D BN-PAGE. Relative quantification of the cytoplasmic membrane proteome by 2D BN-PAGE.

The evolutionary conservation of the phosphoproteomes.a, E. coli. b, B. subtilis. The evolutionary conservation of the phosphoproteomes.a, E. coli. b,

Proteins previously reported in published MALDI IMS studies and their frequency of observation in the present study. Proteins previously reported in published.

Characterization of aggregates isolated from E

Characterization of differentially expressed proteins based on their COG classification. Characterization of differentially expressed proteins based on.

High correlation of expression changes of NMD-regulated genes identified by both the pSILAC screen and previously reported global RNA screens after UPF1.

Analysis of newly synthesized proteins by combined pulsed SILAC and click chemistry enrichment. Analysis of newly synthesized proteins by combined pulsed.

Analysis of whole cell lysates by Western blotting and 2D gel electrophoresis.A, cells overexpressing GFP fusion proteins and control cells were cultured.

Putative targets of miRNAs directly induced by p53 with down-regulated mRNA- and de novo protein synthesis or reduced de novo protein synthesis only. Putative.

Interaction networks of the regulated phosphoproteins.

Differential expression of apoA-I and Vimentin on 2D gels

Bar plot representation of the transcriptomic changes in Δsaci_ptp and Δsaci_pp2a. Bar plot representation of the transcriptomic changes in Δsaci_ptp and.

Ras membrane trap: overview and screen.A, principle of the method.

Localization of selected clones in mammalian COS-7 cells.

Analytical metrics of yeast proteome analysis using the Q-OT-qIT (Fusion) as compared with qIT-OT (Orbitrap Elite) and Q-OT (Q-Exactive) hybrids. Analytical.

Voronoi treemaps (109) comparing protein expression profiles of M

Classification of the 1458 identified proteins into molecular functions. Classification of the 1458 identified proteins into molecular functions. The pie.

Enlargements of 2-D gels visualized by Coomassie Brilliant Blue staining. Enlargements of 2-D gels visualized by Coomassie Brilliant Blue staining. In.

Western blotting analysis of purified cytoplasmic membranes.

Consequences of membrane protein overexpression in E. coli.

Changes in protein expression during distinct stages of NK cell differentiation. Changes in protein expression during distinct stages of NK cell differentiation.

Fur homodimerization and functional assays

Occurrence of fur−-only genes and expected sensitivity to Fur.

Characterization of various mammalian TF-Cub-Baits in yeast

Hijacking the Cell: Parasites in the Driver's Seat

Volume 3, Issue 2, Pages (August 2002)

Ectopic overexpression of WOR3 drives switching to the opaque cell type. Ectopic overexpression of WOR3 drives switching to the opaque cell type. (A) Visualization.

Organelle proteomics in the cardiovascular system.Taylor et al.

Fig. 1 Identification of an alkane synthase in C. variabilis.

USP2a regulates MYC expression through the MDM2–p53 axis.

The N-terminal α-helices of YopM mediate cellular uptake and are able to deliver proteins as cargo. The N-terminal α-helices of YopM mediate cellular uptake.

S. cerevisiae Bre1p confers resistance to apoptosis induced by H2O2.

Decreased KATP activity for SUR1 (ABCC8) channels harboring R1420H or R1420C mutations. Decreased KATP activity for SUR1 (ABCC8) channels harboring R1420H.

Biologic processes necessary for survival after alkylation damage are conserved and when compiled generate a cross-species functionome. Biologic processes.

Research programmes funded by organisations in Africa in 2015.

ERM proteins promote CD44 cleavage.

Proteome changes associated with CHCHD2 expression in NSCLC cells.

Presentation transcript:

Jan-Willem de Gier Center for Biomembrane Research, Stockholm, Sweden Green Light for Membrane Proteins

David Drew et al. Nat Methods (2006) 4:303-13

Monitoring overexpression of a YedZ-GFP fusion using in-gel and whole-cell fluorescence David Drew et al. Nat Methods (2006) 4:303-13

From detergent screen to recovered membrane protein David Drew et al. Nat Methods (2006) 4:303-13

Recovered membrane proteins can be used for functional and structural studies David Drew et al. Protein Sci (2005) 14:

Simon Newstead et al. PNAS (2007) 104(35): GFP-based pipeline in yeast

Biogenesis of membrane proteins in E. coli Samuel Wagner et al. (2006) Trends Biotechnol 24: membrane proteins secretory proteins

E. coli BL21(DE3)/pET overexpression system Merck Novagen, pET manual

Experimental setup (1) Samuel Wagner et al. (2007) Mol Cell Proteomics (9)

Experimental setup (2) Samuel Wagner et al. (2007) Mol Cell Proteomics (9)

Consequences of membrane protein overexpression in E. coli (1) Samuel Wagner et al. (2007) Mol Cell Proteomics (9)

Consequences of membrane protein overexpression in E. coli (2) Samuel Wagner et al. (2007) Mol Cell Proteomics (9)

Characterization of C41(DE3) and C43(DE3), also known as the Walker strains

Differences in overexpression kinetics? Samuel Wagner et al. (2008) PNAS, 105 (38):

Differences in overexpression kinetics

Effect of the addition of IPTG Samuel Wagner et al. (2008) PNAS, 105 (38):

Mutations in the promoter governing T7RNAP expression in C41(DE3) and C43(DE3) Samuel Wagner et al. (2008) PNAS, 105 (38):

Mimicking the effect of the mutations in the promoters with T7 lysozyme Samuel Wagner et al. (2008) PNAS, 105 (38):

Mimicking the effect of the mutations in the promoters with T7 lysozyme Samuel Wagner et al. (2008) PNAS, 105 (38):

Characterization of Lemo21(DE3) Samuel Wagner et al. (2008) PNAS, 105 (38):

Lemo21(DE3) is an ’all in one’ platform for membrane protein overexpression Samuel Wagner et al. (2008) PNAS, 105 (38):

Funding -Swedish Research Council (VR) -Wallenberg Foundation -Swedish Foundation for Strategic Research (SSF) -National Institutes of Health (NIH)